Tripeptide Self-Assembled Monolayers as Biocompatible Surfaces for Cytochrome c Electrochemistry.

Biocompatible tripeptide self-assembled monolayers (SAMs) are designed with a carboxylate group on the terminal amino acid (glutamate, aspartate, or amino adipate) to electrostatically attract the lysine groups around the heme crevice in horse heart cytochrome c (cyt c), creating an electroactive protein/tripeptide/Au interfacial structure. Exposing the peptide/Au electrode to cyt c resulted in an 11 ± 3 pmol/cm2 electroactive protein surface coverage. Topographical images of the interfacial structure are obtained down to single-protein resolution by atomic force microscopy. Uniform protein monolayer assemblies are formed on the Au electrode with no major surface roughness changes. The cyt c/peptide/Au electrode systems were examined electrochemically to probe surface charge effects on the redox thermodynamics and kinetics of cyt c. Neutralization of protein surface charge due to adsorption on anionic COOH-terminated SAMs was found to change the formal potential, as determined by cyclic voltammetry. The cyt c/peptide/Au electrodes exhibit formal potentials shifted to more positive values, have a surface carboxylic acid pKa of 6 or higher, and produce effective cyt c surface charges (Zox) of -6 to -14. The Marcus theory is utilized to determine the protein electron transfer rates, which are ∼5 times faster for cyt c/tripeptide/Au compared to cyt c/11-mercaptoundecanoic acid SAMs of similar chain lengths.

The effect of ionic strength on the electron-transfer rate of surface immobilized cytochrome C.

Horse heart cytochrome c was immobilized on four different self-assembled monolayer (SAM) films. The electron tunneling kinetics were studied in the different assemblies as a function of the ionic strength of the buffer solution using cyclic voltammetry. When cytochrome c is electrostatically immobilized, the standard electron exchange rate constant k0 decreases with the increase of the solution's ionic strength. In contrast, the protein covalently attached or ligated has a rate constant independent of the ionic strength. The inhomogeneity of electrostatically immobilized cytochrome c increases with the increase of the solution's ionic strength whereas that of the covalently attached protein is independent of the ionic strength. A comparison of these different electron-transfer behaviors suggests that the thermodynamically stable geometry of cytochrome c in the electrostatic assemblies is also an electron transfer favorable one. It suggests that the surface charges of cytochrome c are capable of guiding it into geometries in which its front surface faces the electron-transfer partner. The inhomogeneity observed in this study indicates that a distribution of cytochrome c orientations and thus a distribution of electron transfer rate constants exists.

Impact of surface immobilization and solution ionic strength on the formal potential of immobilized cytochrome C.

Four different self-assembled monolayer (SAM) electrode systems were examined electrochemically in order to better understand surface charge effects on the redox thermodynamics of immobilized horse heart cytochrome c (cyt c). Neutralization of protein surface charge upon adsorption on anionic COOH-terminated SAMs was found to cause substantial changes in the formal potential, as determined by cyclic voltammetry. For cyt c immobilized on negatively charged surfaces, the formal potential shifted to more negative values as the ionic strength was decreased, which is opposite to the trend displayed by solution cyt c. In contrast, immobilization to uncharged interfaces resulted in an ionic strength dependence for cyt c that is similar to its solution behavior. The results provide insight into the importance of surface charge on the formal potential of cyt c.